Consistent with the ability of herpesviruses to establish persistent and latent infections, about 25% of all herpesvirus genes modulate and/or abrogate host cellular responses to viral infection. Recently, 140 microRNAs have been identified in a-, b-, and g-herpesvirus genomes which for CMV and KSHV also target host immune functions. MicroRNAs are short 22 + 3 nt RNAs that post-transcriptionally regulate gene expression by binding to 3'UTRs of mRNAs and inducing translational silencing or RNA degradation. To date, only a few genes have been experimentally proven to be miRNA targets. From these limited studies it is clear that viral miRNAs regulate fundamental cellular processes including innate and adaptive immunity, angiogenesis, and apoptosis, and key steps in the herpesvirus life cycle, latency and the switch from latent to lytic replication. Modulation of these processes by microRNAs is likely to affect host/virus interactions and thereby directly contribute to viral pathogenesis. In addition, understanding how miRNAs target innate and adaptive immune function will be critically important for herpesvirus vaccine development. To study the role of human herpesvirus-encoded miRNAs, we propose to establish a core laboratory with the mission to systematically generate a library of herpesvirus genomes that carry single and multiple miRNA mutations. To eliminate the possibility of secondary mutations, recombinant genomes will be re-sequenced using our well established massively parallel sequencing facility. This concerted effort will create highly valuable reagents that will enable studies on how miRNAs function in the context of viral infection, and where appropriate systems exist, in the context of animal models. Importantly, after quality control, each mutant virus will be freely shared with the research community. Creating a core laboratory for this specialized task will have high impact and significantly increase research output in this novel and highly significant field of investigation. Furthermore, in the future, such a core laboratory can be expanded towards targeted mutagenesis of additional genes that can be requested on a for fee basis by investigators at any US research site.